1. Oxidative Phosphorylation
Chapter 19 (Page )

2. 1. Energy from Reduced Fuels is used to Synthesize ATP in Animals
Carbohydrates, lipids, and amino acids are the main reduced fuels for the cell.
Electrons from reduced fuels are transferred to reduced cofactors NADH or FADH2.
In oxidative phosphorylation, energy from NADH and FADH2 are used to make ATP.

3. 2. Energy Flow in Cellular Respiration
Stage 1: Oxidation of metabolic fuels yielding acetyl-CoA.
Stage 2: Oxidation of acetyl groups in the citric acid cycle includes four steps in which electrons are abstracted.
Stage 3: Electrons carried by NADH and FADH2 are funneled into a chain of mitochondrial electron carriers-the respiratory chain-ultimately reducing O2 to H2O.
e- flow drives the production of ATP
FIGURE 16–1 Catabolism of proteins, fats, and carbohydrates in the three stages of cellular respiration. Stage 1: oxidation of fatty acids, glucose, and some amino acids yields acetyl-CoA. Stage 2: oxidation of acetyl groups in the citric acid cycle includes four steps in which electrons are abstracted. Stage 3: electrons carried by NADH and FADH2 are funneled into a chain of mitochondrial (or, in bacteria, plasma membrane–bound) electron carriers—the respiratory chain—ultimately reducing O2 to H2O. This electron flow drives the production of ATP.

4. 3. Oxidative Phosphorylation
Electrons from the reduced cofactors NADH and FADH2 are passed to proteins in the respiratory chain.
In eukaryotes, oxygen is the ultimate electron acceptor for these electrons.
Energy of oxidation is used to phosphorylate ADP generating ATP.
This process was discovered in 1948 by Eugene Kennedy and Albert Lehninger.

5. 3. Oxidative Phosphorylation
FIGURE 16–1 (part 3) Catabolism of proteins, fats, and carbohydrates in the three stages of cellular respiration. Stage 1: oxidation of fatty acids, glucose, and some amino acids yields acetyl-CoA. Stage 2: oxidation of acetyl groups in the citric acid cycle includes four steps in which electrons are abstracted. Stage 3: electrons carried by NADH and FADH2 are funneled into a chain of mitochondrial (or, in bacteria, plasma membrane–bound) electron carriers—the respiratory chain—ultimately reducing O2 to H2O. This electron flow drives the production of ATP.

6. Highly Thermodynamically Unfavorable
3A. Chemiosmotic Theory
ADP + Pi  ATP
Highly Thermodynamically Unfavorable
How do we make it possible?
Phosphorylation of ADP is not a result of a direct reaction between ADP and some high-energy phosphate carrier. Rather the process involves:
Energy needed to phosphorylate ADP is provided by the flow of protons down the electrochemical gradient
The energy released by electron transport is used to transport protons against the electrochemical gradient

7. 3A. Chemiosmotic Theory Four e- carrying complexes.
ATP Synthase Complex
FIGURE 19–1 The chemiosmotic mechanism for ATP synthesis. (a) In mitochondria, electrons move through a chain of membrane-bound carriers (the respiratory chain) spontaneously, driven by the high reduction potential of oxygen and the relatively low reduction potentials of the various reduced substrates (fuels) that undergo oxidation in the mitochondrion. (b) In chloroplasts, the movement of electrons through a chain of membrane-bound carriers is driven by the energy of photons absorbed by the green pigment chlorophyll. In both organelles, electron flow creates an electrochemical potential by the transmembrane movement of protons and positive charge. In both cases this electrochemical potential drives ATP synthesis by a membrane-bound enzyme, ATP synthase, that is fundamentally similar in both mitochondria and chloroplasts, and in bacteria and archaea as well.

8. 3B. Chemiosmotic Energy Coupling Requires Membranes
The proton gradient needed for ATP synthesis can be stably established across a membrane that is impermeable to ions.
Inner membrane in mitochondria
Membrane must contain proteins that couple the energetically “downhill” flow of electrons in the electron-transfer chain with the energetically “uphill” flow of protons across the membrane.
Membrane must contain a protein that couples the “downhill” flow of protons to the phosphorylation of ADP.

9. 4. Structure of a Mitochondrion
Double membrane leads to four distinct compartments:
Outer Membrane:
Relatively porous membrane allows passage of metabolites
Intermembrane Space (IMS):
similar environment to cytosol
higher proton concentration (lower pH)
Inner Membrane
Relatively impermeable, with proton gradient across it
Location of electron transport chain complexes
Convolutions called Cristae serve to increase the surface area
Matrix
Location of the citric acid cycle and parts of lipid and amino acid metabolism
Lower proton concentration (higher pH)

10. 4. Structure of a Mitochondrion
FIGURE 19–2 Biochemical anatomy of a mitochondrion. (a) The outer membrane has pores that make it permeable to small molecules and ions, but not to proteins. The convolutions (cristae) of the inner membrane provide a very large surface area. The inner membrane of a single liver mitochondrion may have more than 10,000 sets of electron-transfer systems (respiratory chains) and ATP synthase molecules, distributed over the membrane surface. (b) The mitochondria of heart muscle, which have more profuse cristae and thus a much larger area of inner membrane, contain more than three times as many sets of electron-transfer systems as (c) liver mitochondria. Muscle and liver mitochondria are about the size of a bacterium—1 to 2 μm long. The mitochondria of invertebrates, plants, and microbial eukaryotes are similar to those shown here, but with much variation in size, shape, and degree of convolution of the inner membrane.

11. 5. Electron-transport Chain Complexes contain a Series of Electron Carriers
Each of the 4 complexes contains multiple redox centers consisting of:
Flavin Mononucleotide (FMN) or Flavin Adenine Dinucleotide (FAD)
Initial electron acceptors for Complex I and Complex II
Can carry two electrons by transferring one at a time
Tightly bound, sometimes covalently, to flavoproteins
Standard reduction potential is dependent on protein environment
Cytochromes a, b or c
Iron-sulfur cluster

12. 5A. Cytochromes One electron carriers (Fe2+ ↔ Fe3+)
Iron coordinating porphoryin ring derivatives
a, b or c are integral proteins but differ by ring additions
c also has peripheral protein form
FIGURE 19–4 Prosthetic groups of cytochromes. (a) Each group consists of four five-membered, nitrogen-containing rings in a cyclic structure called a porphyrin. The four nitrogen atoms are coordinated with a central Fe ion, either Fe2+ or Fe3+. Iron protoporphyrin IX is found in b-type cytochromes and in hemoglobin and myoglobin (see Fig. 4–17). Heme c is covalently bound to the protein of cytochrome c through thioether bonds to two Cys residues. Heme a, found in a-type cytochromes, has a long isoprenoid tail attached to one of the five-membered rings. The conjugated double-bond system (shaded light red) of the porphyrin ring has delocalized π electrons that are relatively easily excited by photons with the wavelengths of visible light, which accounts for the strong absorption by hemes (and related compounds) in the visible region of the spectrum. (b) Absorption spectra of cytochrome c (cyt c) in its oxidized (blue) and reduced (red) forms. The characteristic α, β, and γ bands of the reduced form are labeled.

13. 5 B. Iron-Sulfur Clusters
One electron carriers
Coordinate Fe2+ by cysteines in the protein and inorganic S atoms
Centers defined by inorganic S coordination
Contain equal number of iron and sulfur atoms
Standard reduction potential of the iron varies depending on the center and interaction with protein.
1 Fe
2Fe-2S
4Fe-4S
FIGURE 19–5 Iron-sulfur centers. The Fe-S centers of iron-sulfur proteins may be as simple as (a), with a single Fe ion surrounded by the S atoms of four Cys residues. Other centers include both inorganic and Cys S atoms, as in (b) 2Fe-2S or (c) 4Fe-4S centers. (d) The ferredoxin of the cyanobacterium Anabaena 7120 has one 2Fe-2S center (PDB ID 1FRD); Fe is red, inorganic S is yellow, and the S of Cys is orange. (Note that in these designations only the inorganic S atoms are counted. For example, in the 2Fe-2S center (b), each Fe ion is actually surrounded by four S atoms.) The exact standard reduction potential of the iron in these centers depends on the type of center and its interaction with the associated protein.

14. 6. Ubiquinone (Coenzyme Q)
Ubiquinone is a lipid-soluble conjugated dicarbonyl compound that readily accepts electrons
Upon accepting two electrons, it picks up two protons to give an alcohol, ubiquinol
Ubiquinol can freely diffuse in the membrane, carrying electrons with protons from one side of the membrane to another side
Coenzyme Q is a mobile electron carrier transporting electrons from Complexes I and II to Complex III

15. 6. Ubiquinone (Coenzyme Q)
FIGURE 19–3 Ubiquinone (Q, or coenzyme Q). Complete reduction of ubiquinone requires two electrons and two protons, and occurs in two steps through the semiquinone radical intermediate.

16. 6. Ubiquinone (Coenzyme Q)

17. 7. Free Energy of Electron Transport
Reduction Potential (E)
∆Eo′ = Eo′(e- acceptor) – Eo′(e- donor)
∆Go′ = –nF∆Eo′
For negative G need positive E
E(acceptor) > E(donor)
Electrons are transferred from lower (more negative) to higher (more positive) reduction potential.
Free Energy released is used to pump protons, storing this energy as the electrochemical gradient.
Higher pH  lower reduction potential

18. 7. Free Energy of Electron Transport
Order of e- can be deduced from E’°:
NADH
→ Q
→ Cyt b
→ Cyt c1
→ Cyt c
→ Cyt a
→ Cyt a3
→ O2

19. 8. Composition of Multienzyme Complexes

20. 8A. Flow of Electrons from Biological Fuels into the Electron-Transport Chain
FIGURE 19–8 Path of electrons from NADH, succinate, fatty acyl–CoA, and glycerol 3-phosphate to ubiquinone. Ubiquinone (Q) is the point of entry for electrons derived from reactions in the cytosol, from fatty acid oxidation, and from succinate oxidation (in the citric acid cycle). Electrons from NADH pass through a flavoprotein with the cofactor FMN to a series of Fe-S centers (in Complex I) and then to Q. Electrons from succinate pass through a flavoprotein with the cofactor FAD and several Fe-S centers (in Complex II) on the way to Q. Glycerol 3-phosphate donates electrons to a flavoprotein (glycerol 3-phosphate dehydrogenase) on the outer face of the inner mitochondrial membrane, from which they pass to Q. Acyl-CoA dehydrogenase (the first enzyme of β oxidation) transfers electrons to electron-transferring flavoprotein (ETF), from which they pass to Q via ETF : ubiquinone oxidoreductase.

21. 8B. NADH:ubiquinone oxidoreductase, a.k.a. Complex I
L-shaped complex and starting point for NADH for electron transfer.
One of the largest macro-molecular assemblies in the mammalian cell.
Over 40 different polypeptide chains, encoded by both nuclear and mitochondrial genes.

22. 8B. NADH:ubiquinone oxidoreductase, a.k.a. Complex I
NADH binding site located in the matrix side.
Noncovalently bound flavin mononucleotide (FMN) accepts two electrons from NADH
Several iron-sulfur centers pass one electron at a time toward the ubiquinone binding site

23. 8BI. Complex I- NADH to Ubiquinone
FIGURE 19–9 NADH : ubiquinone oxidoreductase (Complex I). (PDB ID 3M9S) Complex I (the crystal structure from the bacterium Thermus thermophilus is shown) catalyzes the transfer of a hydride ion from NADH to FMN, from which two electrons pass through a series of Fe-S centers to the Fe-S center N-2 in the matrix arm of the complex. Electron transfer from N-2 to ubiquinone on the membrane arm forms QH2, which diffuses into the lipid bilayer. This electron transfer also drives the expulsion from the matrix of four protons per pair of electrons. The detailed mechanism that couples electron and proton transfer in Complex I is not yet known, but probably involves a Q cycle similar to that in Complex III in which QH2 participates twice per electron pair (see Fig. 19–12). Proton flux produces an electrochemical potential across the inner mitochondrial membrane (N side negative, P side positive).
NADH
NAD+ + H+

24. 8BII. NADH:Ubiquinone oxidoreducase is a proton pump
Transfer of two electrons from NADH to ubiquinone is accompanied by a transfer of protons from the matrix (N for loss of protons) to the intermembrane space (P for gain of protons)
Experiments suggest that about four protons are transported per one NADH
NADH + Q + 5H+N → NAD+ + QH2 + 4 H+P
Reduced coenzyme Q picks up two protons
N denotes negative charge due to loss of protons. P denotes positive charge due to gain of protons.
Protons are transported by proton wires
A series of amino acids that undergo protonation and deprotonation to get a net transfer of a proton from one side of a membrane to another

25. 8C. Succinate Dehydrogenase, a.k.a. Complex II
Complex II is the starting point for FADH2 for electron transfer.
FAD accepts two electrons from succinate (recall the citric acid cycle).

26. 8C. Succinate Dehydrogenase, a.k.a. Complex II
Electrons are passed, one at a time, via iron-sulfur centers to ubiquinone, which becomes reduced QH2.
Does not transport protons.

27. 8CI. Complex II- Succinate to Ubiquinone
The complex consists of 4 subunits, two of which (C and D) are transmembrane.
Heme b is not on the main path of e- transfer but may protect against reactive oxygen species.
FIGURE 19–10 Structure of Complex II (succinate dehydrogenase). (PDB ID 1ZOY) This complex (shown here is the porcine heart enzyme) has two transmembrane subunits, C and D; the cytoplasmic extensions contain subunits A and B. Just behind the FAD in subunit A is the binding site for succinate. Subunit B has three Fe-S centers, ubiquinone is bound to subunit B, and heme b is sandwiched between subunits C and D. Two phosphatidylethanolamine molecules are so tightly bound to subunit D that they show up in the crystal structure. Electrons move (blue arrows) from succinate to FAD, then through the three Fe-S centers to ubiquinone. The heme b is not on the main path of electron transfer but protects against the formation of reactive oxygen species (ROS) by electrons that go astray.

28. 8D. Ubiquinone:Cytochrome c Oxidoreductase, a.k.a. Complex III
Uses two electrons from QH2 to reduce two molecules of cytochrome c.
Involves iron-sulfur clusters, cytochrome bs, and cytochrome cs.
Engages in a Q cycle, which results in four additional protons being transported to the intermembrane space.

29. 8DI. Complex III- Ubiquinone to Cyt C
The complex is a dimer of identical monomers, each with 11 different subunits.
The complex has two distinct binding sites for ubiquinone, QN and Qp.
Rieske iron-sulfur proteins have Fe-S clusters with His residue attachments in place of Cys.
FIGURE 19–11 Cytochrome bc1 complex (Complex III). (PDB ID 1BGY) The complex is a dimer of identical monomers, each with 11 different subunits. The functional core of each monomer is three subunits: cytochrome b (green) with its two hemes (bH and bL), the Rieske iron-sulfur protein (purple) with its 2Fe-2S centers, and cytochrome c1 (blue) with its heme. This cartoon view of the complex shows how cytochrome c1 and the Rieske iron-sulfur protein project from the P surface and can interact with cytochrome c (not part of the functional complex) in the intermembrane space. The complex has two distinct binding sites for ubiquinone, QN and QP, which correspond to the sites of inhibition by two drugs that block oxidative phosphorylation. Antimycin A, which blocks electron flow from heme bH to Q, binds at QN, close to heme bH on the N (matrix) side of the membrane. Myxothiazol, which prevents electron flow from QH2 to the Rieske iron-sulfur protein, binds at QP, near the 2Fe-2S center and heme bL on the P side. The dimeric structure is essential to the function of Complex III. The interface between monomers forms two caverns, each containing a QP site from one monomer and a QN site from the other. The ubiquinone intermediates move within these sheltered caverns. Complex III crystallizes in two distinct conformations (not shown). In one, the Rieske Fe-S center is close to its electron acceptor, the heme of cytochrome c1, but relatively distant from cytochrome b and the QH2- binding site at which the Rieske Fe-S center receives electrons. In the other, the Fe-S center has moved away from cytochrome c1 and toward cytochrome b. The Rieske protein is thought to oscillate between these two conformations as it is first reduced, then oxidized.

30. 8DII. The Q Cycle
FIGURE 19–12 The Q cycle, shown in two stages. The path of electrons through Complex III is shown by blue arrows. The movement of various forms of ubiquinone is shown with black arrows. In the first stage (left), Q on the N side is reduced to the semiquinone radical, which moves back into position to accept another electron. In the second stage (right), the semiquinone radical is converted to QH2. Meanwhile, on the P side of the membrane, two molecules of QH2 are oxidized to Q, releasing two protons per Q molecule (four protons in all) into the intermembrane space. Each QH2 donates one electron (via the Rieske Fe-S center) to cytochrome c1, and one electron (via cytochrome b) to a molecule of Q near the N side, reducing it in two steps to QH2. This reduction also consumes two protons per Q, which are taken up from the matrix (N side). Reduced cyt c1 passes electrons one at a time to cyt c, which dissociates and carries electrons to Complex IV.